Rapid and Robust PCR-Based All-Recombinant Cloning Methodology.

ABSTRACT: We report here a PCR-based cloning methodology that requires no post-PCR modifications such as restriction digestion and phosphorylation of the amplified DNA. The advantage of the present method is that it yields only recombinant clones thus eliminating the need for screening. Two DNA amplification reactions by PCR are performed wherein the first reaction amplifies the gene of interest from a source template, and the second reaction fuses it with the designed expression vector fragments. These vector fragments carry the essential elements that are required for the fusion product selection. The entire process can be completed in less than 8 hours. Furthermore, ligation of the amplified DNA by a DNA ligase is not required before transformation, although the procedure yields more number of co

SUBMITTER: Dubey AA 

PROVIDER: S-EPMC4805250 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature